Method of pregnancy diagnosis



United States Patent 3,361,633 METHQD {3F PREGNANQY DIAGNGSBS KurtLange-Sundermann, Bremen, Germany, assignor to Martin BriniunannAlrtiengeseiischaft, Zweigniederiassung, Berlin, Germany No Drawing.Filed Sept. 16, 1965, Ser. No. 487,9{37 Claims priority, applicationGermany, July 1, 1961,

Sch 29,937

9 Claims. (Cl. 167-845) The present application is acontinuation-in-part of my copending application Ser. No. 207,018, filedJuly 2, 1962, and entitled Quick Acting Agent Against Amenorrhea, nowPatent No. 3,236,731, granted Feb. 22, 1966.

My above mentioned copending application discloses a new series ofsteroid compounds which are not only valuable agents in the treatment ofamenorrhea and glandular cystic hyperplasia, but which also may servefor the early diagnosis of pregnancy.

It is an object of the present invention to provide a method ofpregnancy diagnosis utilizing these new steroid compounds, which methodis simple and capable of diagnosing pregnancy at a very early stage.

Other objects and advantages of the present invention will be apparentfrom a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainlycomprises administering to a female patient for the purpose ofdetermining whether or not the patient is pregnant a compound selectedfrom the group consisting of compounds of the formula:

RCH CH2 and compounds of the formula:

CH3 CH3 RCH wherein R is selected from the group consisting of fi-OH andS-Oacyl, wherein R is selected from the group consisting of S-OH,B-Oacyl and :O, and wherein R" is selected from the group consisting offi-OH and B-Oacyl, acyl in all cases being derived from a loweraliphatic carboxylic acid.

It has been found in accordance with the present invention that the newA -pregnene-6B,20diol-3-one, as well as the new isomericallopregnane-20-ol-3,6dione which is formed from A-pregnene-6B,2Ofi--diol-3-one by intramolecular rearrangement thereof,as well as the esters of these compounds, are active agents with theabove mentioned desired characteristics. Thus these compounds cause thedesired menstrual bleeding of the uterus mucosa already within a fewhours after administration thereof.

3,361,633 Patented Jan. 2, 1968 Although the 6-position hydroxyl groupof the A -pregnene-6,20,B-diol-3-one is in fi-position, the question ofwhether it is in alpha or beta position is of little importance becauseafter the administration of the A -pregnene compound into the humanorganism it rearranges by biochemical reaction into the isomericallopregnane compound, which has the same action as the A -pregnenecompound.

Until now only suppositions (capillary effect actionsee further below)had been made concerning the detailed mechanism of action which causesthe start of menstrual bleeding of the uterus mucosa afteradministration of the new agents of the present invention. One hint isgiven by the observation that in the presence of progesterone, eitherdue to a condition of pregnancy or due to prior or simultaneousadministration of progesterone, the bleeding-causing eifect of the newagents of the present invention does not occur.

For this reason it is apparent that the new agents are Well suited forthe early diagnosis of pregnancy.

The above mentioned antagonism between the new agents and theprogestational agents leads to the assumption that possibly with the newagents of the present invention there has been found the actualmenstruationcausing hormones, the existence of which have been suspectedfor quite some time. It would be expected that the menstruation causinghormones, the action of which can be suppressed by progestationalagents, would influence the juvenile capillary vessels of the uterusmucosa so as to cause bleeding.

Pregnancy diagnostic tests were conducted on seven women ranging from 21to 40 years of age, whose menses were overdue. Each woman wasintravenously administred 50 milligrams of the ZO-hemisuccinate ester ofallopregnane-ZO-beta-ol-3,6-dione. No bleeding occurred in any womantested. Pregnancy was confirmed in each case, generally by pregnancytests carried out on frogs.

Generally intravenous administration of 50 milligrams represents adesired dosage. Fifty milligrams, generally, are also the minimum dosagerequired for the intended purpose. However, the pregnancy test is notlimited to administration of 50 milligrams of the diagnostic agent usedaccording to the present invention and, depending on the specificcircumstances, other dosages, generally within the range of bet-ween 50milligrams and milligrams may be used.

The compounds used according to the present invention may be producedusing methods which aside from the materials being treated are otherwiseanalogous methods in the art of steroid chemistry.

A preferred starting material is the hitherto unknown compound A-pregnene-3fi,6,8,20,B-triol which is easily obtainable from the known A-pregnene-3fi,6/3-diol-20-one by reduction by means of lithium aluminumhydride, sodium borohydride or other suitable reduction agents.

By the selective oxidation of the 3-position hydroxyl group of A-pregnene-3B,6,8,2Ofi-triol utilizing known methods of oxidation,preferably by the use of manganese dioxide in a solvent which is inertto the oxidation agent, the A -pregnene-6B,20;3-diol-3-one is obtained.At the same time there is obtained, because of partial rearrangement ofthe primary oxidation product, a considerably amount ofallopregnane-20fl-ol-3,6-diol. By recrystallization of the crudeoxidation product the produced A*-pregnene-6;8,20B- diol-3-one is onlypartially (about 20%) isolated in pure condition.

If desired the pure A -pregnene-6 8,20 8-diol-3-one can subsequently inknown manner be converted into its 6B derivative and/or ZOE-derivative,particularly into the esters of lower aliphatic carboxylic acids e.g.acetate, propionate, butyrate and succinate, or of suitable anorganicacids e.g. hemisulfate and phosphate which are most useful in thesteroid chemistry field. These derivatives can be used as pharmaceuticalagents.

In addition, the intermediate production of such derivatives can also beused for the further purification of the crudeoxidation product. Thepure A*-pregnene-6,8,20B- 'diol-3-one by heating in alkaline or acidmedium can be practically quantitatively converted to allopregnane-ZOB-ol-3,6-dione. The pure production of the allopregnane-ZQB- ol-3,6-dioneis preferable either after separation of the pure A-pregnene-6fi,20fi-diol 3 one remaining syrupy crystallization residueor the entire mass of crude product obtained by the selective oxidationwith manganese dioxide can be rearranged with alkali and the pureallopregnane-fl-ol-3-dione can then in normal manner, for example bycrystallization from the crude rearrangement product, be separated. Ifdesired the separated allopregnane-20,8-ol-3,6-dione can subsequently inknown manner be converted into its 20,8-derivative, particularly into anester such as a lower aliphatic carboxylic acid ester, which is usefulin the art of steroid chemistry.

The following examples are given to further illustrate the presentinvention. The scope of the invention is not, however, meant to belimited to thespecific details of the examples.

EXAMPLE 1 (a) Production of the starting material 20 g. of A-pregnene-35,6/3-diol-20-one are allowed to stand at room temperaturefor one hour with 1.0 g. of sodium borohydride in 180 cc. of 80% aqueousdioxane. The excess of sodium borohydride is subsequently mixed with 50cc. of 20% acetic acid and with water is then diluted to a volume of 500cc. The aqueous solution is extracted three times with chloroform, thepurified chloroform phase is washed three times, each time with 300 cc.of water, and dried over water-free sodium sulfate.

After filtering off the drying agent the chloroform is evaporated undervacuum to dryness. The residue of 18.9 g. (94. 5% of the theoretical) isrecrystallized two times from acetone-petroleum ether (boiling point7090 C.), whereby the melting point of the obtained A -pregnene-3j3,6,8,20,8-triol is increased to 185-185 .5 C. The product is convertedinto the triacetate for further characterization. The melting point isl71.5173 C. [a] =5.6.

(b) Oxidation of the starting material 2.0 g. of A-pregnene-3[3,65,20B-triol are shaken at room temperature for hours with20 g. of manganese dioxide in 200 cc. of chloroform. After standing theoverlying chloroform solution is filtered off and the manganese dioxideresidue is cooked five times, each time with 100 cc. of chloroform. Thepurified chloroform extract is evaporated to dryness under vacum. Theevaporation residue of 1.4 g. (70% of the theoretical), is dissolved inacetone and mixed with petroleum ether (boiling point 7090 C.) untilturbidity. After following crystallization the obtained 435 mg. (21.8%)of A -pregnene-diol-613,20fl-3-one having a melting point of 18l.5185 C.is recrystallized two times from acetone-petroleum ether (boiling point70- 90 C.) resulting in an increase in the melting point to l86.5189 C.i 1680 cm."

The residue which separates as a syrup contains considerable amounts ofallopregnane-20B-ol-3,6-dione besides additionalM-pregnene-6,3,20fi-diol-3-one.

EXAMPLE 2 Rearrangement of pure A -pregnene-6B,20fl-di0l-3-0ne Thesolution of mg. of A -pregnene-3one-63,20,3- diol in 10 cc. of methanolis mixed with 0.5 g. of potassium hydroxide dissolved in 2 cc. of Waterand cooked under refluxing for one hour. After cooling the solution isneutralized with dilute acetic acid, extracted three times withchloroform, the purified chloroform extract washed with water, driedwith water-free sodium sulfate and after filtration of the solvent isbrought to dryness under vacuum by evaporation. The thus obtained crudeallopregnane- 20/3-ol-3,6dione, obtained in a yield of 41.3 mg. (82.6%of the theoretical) is recrystallized two times from acetone-petroleumether (boiling point 7 0-90 C.) and is then found to have a meltingpoint of 232-236 C.

)t =l7l4 cm.- EXAMPLE 3 Rearrangement of the crystallization residue 1.2g. of syrupy crystallization residue obtained according to Example 1(b)is dissolved in 200 cc. of methanol, mixed with 1.4 g. of potassiumhydroxide in 40 cc. of water and cooked under refluxing for one hour.Thereafter the reaction mixture is further worked up analogous- :ly toExample 2 and there is thus obtained an additional 0.765 g. (68% of thetheoretical) of allopregnane-ZOB-ol- 3,6-dione.

EXAMPLE 4 g 2.0 g. of 5a-pregnane-20fi-ol-3,6-dione with 3.0 g. ofsuccinic acid anhydride, 1.0 g. of succinic acid and 8.0 cc. of pyridineare heated to refluxing for 3 /2 hours. Subsequently the reactionmixture is poured into ice water under stirring, the separated productis filtered ofl under suction, washed under water and dried. The thusobtained 20-hemisuccinate of 5a-pregnane-20fi-ol-3,6- dione afterrecrystallization from ethyl acetate melts at 227.5230 C. The yield is2.3 g. Further recrystallization results in increase of the meltingpoint to 228.5-230 C. [a] =19 (CHClg).

The hemisuccinate dissolves in water with sodium carbonate with theformation of the hemisuccinate-sodium salt. Analogously there isobtained from the hemisuccinate with methyl glucamine the methylglucamine salt.

EXAMPLE 5 5.0 g. of 5a-pregnane-20t3-ol-3,6-dione are heated four hourson a steam bath with 12 cc. of propionic acid anhydride in 20 cc. ofpyridine. The reaction product is then stirred into ice water and thestirring is continued for an additional two hours. After the precipitateis filtered off under suction it is washed with water, dried and thenrecrystallized from methanol. The thus obtained 20-propionate of5a-pregnane-20r3-ol-3,6-dione melts at 197- 199 C. After furtherrecrystallization the substance exhibits a melting point of 200-200.5 C.[a] =18 (CHCl Without further analysis, the foregoing will so fullyreveal the gist of the present invention that others can by applyingcurrent knowledge readily adapt it for various applications withoutomitting features that, from the standpoint of prior art, fairlyconstitute essential characteristics of the generic or specific aspectsof this inyention and, therefore, such adaptations shoud and areintended to be comprehended within the meaning and range of equivalenceof the following claims What is claimed as new and desired to be securedby Letters Patent is:

1. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount of a compound selectedfrom the group consisting of compounds of the formula:

and compounds of the formula:

CH3 RH wherein R is selected from the group consisting of B-OH andB-Oacyl, wherein R is selected from the group consisting of fl-OH,fi-Oacyl and :0, and wherein R" is selected from the group consisting ofB-OH and ,B-Oacyl, Oacyl in all cases being selected from the groupconsisting of acetate, propionate, butyrate, succinate, hemisulfate andphosphate, whereby in a nonpregnant patient menstrual bleeding is causedwhile in a pregnant patient no such bleeding occurs.

2. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount of A-pregnene-6p,20/8-diol-3-one, whereby in a nonpregnant patient menstrualbleeding is caused while in a pregnant patient no such bleeding occurs.

3. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an etiective amount of A-pregnene-6fi,20/3-diol-3-one-20-ester wherein ester is selected fromthe group consisting of acetate, propionate, butyrate, succinate,hemisulfate and phosphate, whereby in a nonpregnant patient menstrualbleeding is caused while in a pregnant patient no such bleeding occurs.

4. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount of A-pregnene-6fi,2Opdiol-3-one-2O-acetate,

6 whereby in a nonpregnant patient menstrual bleeding is caused while ina pregnant patient no such bleeding occurs.

5. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount ofallopregnane-20/3-ol-3,6-dione, whereby in a nonpregnant patientmenstrual bleeding is caused While in a pregnant patient no suchbleeding occurs.

6. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount ofallopregnane-2OB-ol-3,6-dione-20-ester wherein ester is selected fromthe group consisting of acetate, propionate, butyrate, succinate,hemisulfate and phosphate, whereby in a nonpregnant patient menstrualbleeding is caused while in a pregnant patient no such bleeding occurs.

7. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an efiective amount ofallopregnane-20fl-ol-3,6-dione-20-acetate, whereby in a nonpregnantpatient menstrual bleeding is caused while in a pregnant patient no suchbleeding occurs.

8. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an effective amount of ZO-hemisuccinate ofSaregnane-ZOB-ol- 3,6-dione, whereby in a nonpregnant patient menstrualbleeding is caused while in a pregnant patient no such bleeding occurs.

59. Method of pregnancy diagnosis, which comprises administering to apotentially pregnant patient an eifective amount 20-propionate of5a-pregnane-2013-ol-3,6- dione, whereby in a nonpregnant patientmenstrual bleeding is caused while in a pregnant patient no suchbleeding occurs.

No references cited.

ALBERT T. MEYERS, Primary Examiner.

SHELDON I. SINGER, Assistant Examiner.

1. A METHOD OF PREGNANCY DIAGNOSIS, WHICH COMPRISES ADMINISTERING TO APOTENTIALLY PREGNANT PATIENT AN EFFECTIVE AMOUNT OF A COMPOUND SELECTEDFROMTHE GROUP CONSISTING OF COMPOUNDS OF THE FORMULA;